<i>C</i>. <i>albicans</i> induced TNF and IL-6 expression in neonatal and adult monocytes.

<p>CBMO and PBMO were infected with <i>C</i>. <i>albicans</i> at a MOI of 5 for 4h, or were treated with the indicated TLR agonists. The role of TLR2 for intracellular TNF-α expression was determined by administration of a TLR2 bAb (A, n = 5; 1-way ANOVA solid/dashed crotched lines, **p<0.01; 2-way-ANOVA blunt-ended lines and stars within bars, *p<0.05;**p<0.01; ***p<0.005). IL-6 secretion 4 h p.i. with or without TLR2 bAb administration as indicated (B, n = 3; student`s t-test solid/dashed crotched lines, **p<0.01; 2-way-ANOVA blunt-ended lines,*p<0.05; **p<0.01).</p

TLR2 and TLR4 surface expression by neonatal or adult monocytes following infection with <i>C</i>. <i>albicans</i> or TLR2 stimulation.

<p>CD14⁺ CBMO and PBMO were infected with <i>C</i>. <i>albicans</i> at a MOI of 5 for 2h (A, C), or treated with indicated TLR2 agonists (B, D). The surface expression of TLR2 (A, B) and TLR4 (C, D) were assessed by flow cytometric analysis (n = 8; 1-way-ANOVA test marked by crotched solid (between PBMO groups) and dotted (between CBMO groups) lines, *p<0.05; **p<0.01, ***p<0,005; 2-way ANOVA test marked by blunt ended lines and asterisks in chart bars testing groups in C and D; **p<0.01).</p

Phagocytic properties of neonatal and adult monocytes.

<p>Comparative flow cytometric analysis of the PI and PC of gated CD14⁺ monocytes in CBMO and PBMO following infection with GFP⁺ <i>C</i>. <i>albicans</i> for 2h at the indicated MOI (n = 3; student`s t-test, * p < 0.05).</p

<i>C</i>. <i>albicans</i> infection-induced caspase-8.

<p>Mean fluorescence intensity (MFI) of the intracellular staining of cleaved caspase-8 in CD14⁺ PBMO (A) or CBMO (B) following infection with <i>C</i>. <i>albicans</i> at a MOI of 1:5. Pre-treatment with TLR2 bAb or MyD88i peptide (n = 5; student`s-t-test marked by solid (PBMO groups) or crotched (CBMO groups) lines, *p<0.05, **p<0.01, ***p<0.005; 1-way_ANOVA marked by blunt ended lines, *p<0.05, **p<0.01, ***p<0.005; 2-way-ANOVA marked by asterisks in chart bars, **p<0.01).</p

<i>C</i>. <i>albicans</i>-induced monocyte apoptosis.

<p><i>C</i>. <i>albicans</i>-induced monocyte apoptosis.</p

The influence of MyD88-dependent innate immune signaling on intraepithelial microcolony formation.

<p>1-day-old MyD88^+/+ and MyD88^-/- mice were orally infected with 100 CFU <i>S</i>. Typhimurium WT. <b>(A)</b> 4 days after infection, small intestinal tissues were collected and analyzed by immunostaining. Three representative images showing <i>S</i>. Typhimurium (red) forming intraepithelial microcolonies in MyD88^-/- mice. Counterstaining with E-cadherin (green), WGA (white) and DAPI (blue). Bar, 10 μm. For wild type controls see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.g001" target="_blank">Fig 1D</a>. <b>(B)</b> Quantitative evaluation of the number of intraepithelial microcolonies per villus in MyD88^+/+ and MyD88^-/- mice at 4 days p.i.. <i>S</i>. Typhimurium microcolonies were quantified in 20–30 villi per animal (n = 6–8). Results represent the mean ± SD. <b>(C)</b> Transmission electron microscopy (TEM) images of intraepithelial <i>Salmonella</i> in MyD88^+/+ (left panel) and MyD88^-/- mice (right panel). Asterisks highlight bacteria. Bar, 2 μm. <b>(D)</b> Co-immunostaining for <i>Salmonella</i> (green) and LAMP1 (red) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(E)</b> Quantitative evaluation of the percentage of intraepithelial <i>S</i>. Typhimurium associated with LAMP1 staining. Four neonates were analyzed at day 4 p.i.. Results represent the mean ± SD. <b>(F)</b> Co-immumostaining for <i>Salmonella</i> (red) and the GFP expressing SPI2 reporter (pM973, green) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(G)</b> Quantitative analysis of the percentage of intraepithelial <i>S</i>. Typhimurium expressing the SPI2 reporter. Microcolonies from tissue sections from four neonates were analyzed at day 4 p.i.. Results represent the mean ± SD.</p

The role of SopB in the interaction between <i>Salmonella</i> and the epithelium.

<p>1-day-old C57BL/6 mice were orally infected with 100 CFU wild type (WT) (filled circles), isogenic <i>sopB</i> mutant (filled triangles), or p<i>sopB</i>-complemented Δ<i>sopB</i> (open triangles) <i>S</i>. Typhimurium. Viable counts in <b>(A)</b> isolated gentamicin-treated enterocytes, <b>(B)</b> total MLN and <b>(C)</b> total liver tissue homogenate at 2 days p.i.. <b>(D)</b> Quantitative RT-PCR for <i>Cxcl2</i> mRNA in total RNA prepared from enterocytes isolated at 2 days p.i.. Values were normalized to uninfected age-matched control animals (crosses). Individual values and the mean from at least two independent experiments are shown (n = 3–5 animals per group). <b>(E)</b> Quantitative analysis of the number of cleaved caspase 3- and <b>(F)</b> cleaved caspase 8 positive cells per 200 times magnification image field. Positive cells from 20 image fields from one section were analyzed per infected neonate (n = 3–6) at day 3 p.i.. Results represent the mean ± SD. <b>(G)</b> Immunostaining for <i>S</i>. Typhimurium (red) in small intestinal tissue sections at 3 days p.i. with 100 CFU WT and Δ<i>sopB S</i>. Typhimurium. Counterstaining with E-cadherin (green), WGA (white) and DAPI (blue). Bar, 5 μm. <b>(H)</b> Co-immunostaining for Δ<i>sopB S</i>. Typhimurium (green) and LAMP1 (red) in small intestinal tissue sections at day 3 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(I)</b> Quantitative evaluation of the percentage of intraepithelial <i>S</i>. Typhimurium associated with LAMP1 staining. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3) at day 3 p.i.. Results represent the mean ± SD. <b>(J)</b> Co-immumostaining for Δ<i>sopB S</i>. Typhimurium (red) and the GFP expressing SPI2 reporter (pM973; green) in small intestinal tissue sections at day 3 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(K)</b> Quantitative analysis of the percentage of intraepithelial <i>S</i>. Typhimurium expressing the SPI2 reporter. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3) at day 3 p.i.. Results represent the mean ± SD.</p

Analysis of <i>sopBE</i>_<i>2</i> and <i>sopAE</i>_<i>2</i> double mutant <i>S</i>. Typhimurium.

<p><b>(A-C)</b> 1-day-old C57BL/6 mice were orally infected with 100 CFU wild type (WT) (filled circles), isogenic Δ<i>sopBE</i>_<i>2</i> (inverted open triangles), or Δ<i>sopAE</i>_<i>2</i> (open triangles) <i>S</i>. Typhimurium. Viable counts in <b>(A)</b> isolated gentamicin-treated enterocytes and <b>(B)</b> total liver tissue homogenate at 4 days p.i.. <b>(C)</b> Quantitative RT-PCR for <i>Cxcl2</i> mRNA in total RNA prepared from enterocytes isolated at 4 days p.i.. Values were normalized to uninfected age-matched control animals (crosses). Individual values and the mean from at least two independent experiments are shown (n = 3–6 animals per group). The data for uninfected control animals and <i>Salmonella</i> WT infected mice are identical to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.g001" target="_blank">Fig 1A–1C</a>. <b>(D)</b> Immunostaining for <i>Salmonella</i> (red) in small intestinal tissue sections at 4 days p.i. with 100 CFU Δ<i>sopE</i>_<i>2</i>, Δ<i>sopBE</i>_<i>2</i>, or Δ<i>sopAE</i>_<i>2</i> <i>S</i>. Typhimurium. Counterstaining with E-cadherin (green), WGA (white) and DAPI (blue). Bar, 5 μm. <b>(E)</b> Percentage of epithelial cells positive for single bacteria or microcolonies (>1 intraepithelial bacterium) at 4 days p.i. with Δ<i>sopBE</i>_<i>2</i> or Δ<i>sopAE</i>_<i>2</i> <i>S</i>. Typhimurium. 30 <i>Salmonella-</i>positive epithelial cells per infected neonate (n = 8–13) were analyzed. Results represent the mean ± SD. <b>(F)</b> Co-immunostaining for <i>Salmonella</i> Δ<i>sopBE</i>_<i>2</i> and Δ<i>sopAE</i>_<i>2</i> (green) and LAMP1 (red) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(G)</b> Quantitative evaluation of the percentage of intraepithelial Δ<i>sopBE</i>_<i>2</i> and Δ<i>sopAE</i>_<i>2</i> <i>S</i>. Typhimurium associated with LAMP1 staining. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3–4) at day 4 p.i.. Results represent the mean ± SD. <b>(H)</b> Co-immunostaining for Δ<i>sopBE</i>_<i>2</i> and Δ<i>sopAE</i>_<i>2</i> <i>Salmonella</i> (red) and the GFP-expressing SPI2 reporter (pM973; green) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(I)</b> Quantitative analysis of the percentage of intraepithelial <i>S</i>. Typhimurium expressing the SPI2 reporter. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3–4) at day 4 p.i.. Results represent the mean ± SD.</p

The redundant role of SipA and SopE₂ for enterocyte invasion.

<p><b>(A-C)</b> 1-day-old C57BL/6 mice were orally infected with 100 CFU wild type (WT) (filled circles) or isogenic <i>sopE</i>_<i>2</i><i>sipA</i> mutant <i>S</i>. Typhimurium (filled triangles). Viable counts in <b>(A)</b> isolated gentamicin-treated enterocytes and <b>(B)</b> total liver tissue homogenate at 4 days p.i.. <b>(C)</b> Quantitative RT-PCR for <i>Cxcl2</i> mRNA in total RNA prepared from enterocytes isolated at 4 days p.i.. Values were normalized to uninfected age-matched control animals (crosses). Individual values and the mean from at least two independent experiments are shown (n = 4–6 animals per group). The data for uninfected control animals and WT <i>Salmonella</i> infected mice are identical to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.g001" target="_blank">Fig 1A–1C</a>. <b>(D-F)</b> 1-day-old C57BL/6 mice were orally infected with 100 CFU WT (filled circles), isogenic <i>sipA</i> mutant (open squares), complemented Δ<i>sipA</i> p<i>sipA</i> (filled squares), isogenic <i>sopE</i>_<i>2</i> mutant (open diamonds), or complemented Δ<i>sopE</i>_<i>2</i> p<i>sopE</i>_<i>2</i> (filled diamonds) <i>Salmonella</i>. Viable counts in <b>(D)</b> isolated gentamicin-treated enterocytes and <b>(E)</b> total liver tissue homogenate at 4 days p.i.. <b>(F)</b> Quantitative RT-PCR for <i>Cxcl2</i> mRNA in total RNA prepared from enterocytes isolated at 4 days p.i.. Values were normalized to uninfected age-matched control animals (crosses). Individual values and the mean from at least two independent experiments are shown (n = 3–5 animals per group). The data for uninfected control animals and <i>Salmonella</i> WT infected mice are identical to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.g001" target="_blank">Fig 1A–1C</a>. <b>(G)</b> Immunostaining for <i>S</i>. Typhimurium (red) in small intestinal tissue sections at 4 days p.i. with 100 CFU Δ<i>sopE</i>_<i>2</i><i>sipA</i>, Δ<i>sipA</i>, or Δs<i>opE</i>_<i>2</i> <i>S</i>. Typhimurium <i>Salmonella</i>. Counterstaining with E-cadherin (green), WGA (white) and DAPI (blue). Bar, 5 μm. <b>(H)</b> Co-immunostaining for <i>Salmonella</i> Δ<i>sipA</i>, Δ<i>sopE</i>_<i>2</i> (green) and LAMP1 (red) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(I)</b> Quantitative evaluation of the percentage of intraepithelial <i>S</i>. Typhimurium associated with LAMP1 staining. All microcolonies from three tissue sections per infected neonate were analyzed (n = 5) at day 4 p.i.. Results represent the mean ± SD. <b>(J)</b> Co-immunostaining for <i>Salmonella</i> Δ<i>sipA</i> or Δ<i>sopE</i>_<i>2</i> (red) and the GFP-expressing SPI2 reporter (pM973; green) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5μm. <b>(K)</b> Quantitative analysis of the percentage of intraepithelial <i>S</i>. Typhimurium expressing the SPI2 reporter. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3) at day 4 p.i.. Results represent the mean ± SD.</p

The role of SipA for intraepithelial microcolony formation.

<p><b>(A)</b> Mucosal barrier integrity tested by serum quantification 4 hours after oral administration of FITC labeled-4kDa dextran. 1-day-old C57BL/6 mice were left untreated (crosses) or infected with WT (filled circles) or Δ<i>sopABE</i>_<i>2</i> (open squares) <i>S</i>. Typhimurium. FITC labeled-4 kDa dextran was quantified in serum at day 3 p.i. as indicated. <b>(B and C)</b> Flow cytometric analysis of lamina propria immune cells. 4-day-old mice were left untreated or orally infected with 100 CFU WT or Δ<i>sopABE</i>_<i>2</i> <i>S</i>. Typhimurium and total small intestinal leukocytes were analyzed by flow cytometry at day 3 p.i.. <b>(B)</b> Monocytes (Ly6C^hiLy6G^-CD11b⁺ MHCII^lo/-CD45⁺DAPI^-) and <b>(C)</b> neutrophils (Ly6G⁺Ly6C^intCD11b⁺ MHCII^lo/-CD45⁺DAPI^-) are depicted as percentage of CD45⁺ cells in non-infected (crosses), WT (filled circles) and Δ<i>sopABE</i>_<i>2</i> <i>Salmonella</i> (open squares). The results represent the mean values from at least two independent experiments (n = 4–6 per group). <b>(D)</b> Immunostaining for <i>Salmonella</i> in small intestinal tissue sections at 4 days after co-infection with 100 CFU GFP-expressing WT (yellow) and Δ<i>sopABsipA</i> (red) <i>S</i>. Typhimurium. WT <i>Salmonella</i> appear in yellow due to simultaneous staining for O4/O5 antigen (red) and GFP (green). Δ<i>sopABsipA Salmonella</i> appear in red due to simultaneous staining for O4/O5 antigen (red). Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 10 μm. <b>(E-J)</b> 1-day-old C57BL/6 mice were orally infected with 100 CFU WT (filled circles), Δ<i>sipA</i> (open diamonds), Δ<i>sipA</i> complemented with p<i>sipA</i>^{K635A E637W} (half-filled diamonds), Δ<i>sipA</i> complemented with p<i>sipA</i>^D434A (filled squares), or Δ<i>sipA</i> complemented with p<i>sipA</i>^{D434A K635A E637W} (filled triangles) <i>S</i>. Typhimurium. Viable counts in <b>(E)</b> isolated gentamicin-treated enterocytes and <b>(F)</b> total liver tissue homogenate at 4 days p.i.. <b>(G)</b> Quantitative RT-PCR for <i>Cxcl2</i> mRNA in total RNA prepared from enterocytes isolated at 4 days p.i.. Values were normalized to uninfected age-matched control animals (crosses). Individual values and the mean from at least two independent experiments are shown (n = 4–7 animals per group). The data for WT <i>Salmonella</i> infected mice and uninfected control animals are identical to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006925#ppat.1006925.g001" target="_blank">Fig 1A–1C</a>. <b>(H)</b> Immunostaining for <i>Salmonella</i> (red) in small intestinal tissue sections at 4 days p.i. with 100 CFU Δ<i>sipA</i>, Δ<i>sipA</i> complemented with p<i>sipA</i>^{K635A E637W}, Δ<i>sipA</i> complemented with p<i>sipA</i>^D434A, or Δ<i>sipA</i> complemented with p<i>sipA</i>^{D434A K635A E637W} <i>S</i>. Typhimurium. Counterstaining with E-cadherin (green), WGA (white) and DAPI (blue). Bar, 5 μm. <b>(I)</b> Co-immunostaining for LAMP1 (red) and Δ<i>sipA</i> complemented with p<i>sipA</i>^{K635A E637W}, Δ<i>sipA</i> complemented with p<i>sipA</i>^D434A, or Δ<i>sipA</i> complemented with p<i>sipA</i>^{D434A K635A E637W} <i>S</i>. Typhimurium (green) in small intestinal tissue sections at day 4 p.i.. Counterstaining with E-cadherin (white) and DAPI (blue). Bar, 5 μm. <b>(J)</b> Quantitative evaluation of the percentage of intraepithelial <i>S</i>. Typhimurium associated with LAMP1 staining. All microcolonies from three tissue sections per infected neonate were analyzed (n = 3) at day 4 p.i.. Results represent the mean ± SD.</p